Enormous efforts have been made to develop circulation systems for physiological nutrient supply and waste removal of in vitro cultured tissues. These developments are aiming for in vitro generation of organ equivalents such as liver, lymph nodes (Giese et al., 2010, Journal of Biotechnology 148, 38-45) and lung (Huh et al., 2010, Science, (328) 5986, pp. 1662-1668) or even multi-organ systems (Sonntag et al, 2010, Journal of Biotechnology 148, 70-75) for substance testing, research on organ regeneration or for transplant manufacturing. Initially technical perfusion systems based on membranes, hollow fibres (Catapano and Gerlach. Bioreactors for Liver Tissue Engineering. 2. Topics in Tissue Engineering, Vol. 3, 2007. 1-42 Eds. N Ashammakhi, R Reis & E Chiellini) or networks of micro channels (Du et al., Chapter 7: Microfluidic Systems for Engineering Vascularized Tissue Constructs”. 2008 (Book chapter)) were used for these purposes.
In a liver support system developed by Gerlach and co-workers three bundles of hollow-fibres are cross-woven with each other, forming multiple identical micro culture spaces for plasma perfusion and oxygen supply. Human plasma perfusion is assured by two micro-filtration hollow fibre membrane bundles, whereas oxygenation takes place through a liquid-impermeable oxygen transport membrane. Liver support systems based on this principle have well-performed over several weeks, being included in a plasma flow circuit of patients.
Du et al. summarised fluidic platforms for generating micro-vascularised tissue constructs on the basis of hydrogels and micro-fabrication techniques. The overview highlights the technical ability to form blood-capillary-network like channel structures within polymeric materials for efficient liquid perfusion through tissue cultures. The majority of the resulting micro systems were used for highly efficient technical perfusion not including endothelial cells.
Such systems are limited to the use of culture media or plasma, but do not allow for whole blood perfusion due to clotting phenomena. In addition they do not provide the natural blood tissue barrier, which in vivo is composed by closed endothelial cell layer. This allows for active transport through the cell layer, as well as for signalling from the tissue into the capillary network. Different approaches were developed to line technical perfusion systems (Song et al., 2005, Anal. Chem., 2005, 77, 3993-3999) or synthetic or biological matrices (Zhang et al., 2009, Biomaterials, 30(19): 3213-3223; Walles, 2010, Journal of Biotechnology 148, 56-63) up with endothelial cells.
Song and co-workers developed a micro circulatory support to culture endothelial cells under defined shear stresses. Closed monolayers of endothelial cells could be established in individual cell culture compartments, located between technical transport channels.
To generate tissue-engineered vascular grafts (TGVG) Zhang et al. developed a tissue engineered construct that mimicked the structure of blood vessels using tubular electrospun silk fibroin scaffolds with suitable mechanical properties. They seeded human coronary artery smooth muscle cells (HCASMCs) and human aortic endothelial cells (HAECs) onto the luminal surface of the tubular scaffolds and cultivated under physiological pulsatile flow, which was generated within the dual loop bioreactor using external tubing and pumps.
A fully biological matrix for the establishment of an endothelialised vasculature in vitro was used by Walles et al., connecting polymeric tubing and controlled pumping system to an acellularised animal gut segment. In this system the capillaries which are entirely covered by endothelial cells are limited to the functionally relevant areas of the biomatrix.
However, none of the prior art circulation systems was suitable for long-term tissue culture based on whole blood as provided in this application.
The present invention relates to a closed and self-contained circulation system emulating the natural blood perfusion environment of vertebrates at tissue level. The system uses a miniaturised physiological blood circulation to provide circulation of micro liter volume to support milligrams of tissue. This mimics the physiological ratio of humans, where liters of blood-volume support kilograms of tissue at a chip-compatible micro scale. The self-contained circulation system contains at least one capillary growth section located between the micro inlets and micro outlets of the system.
A capillary growth section for formation of blood capillaries, supporting nutrient exchange, is integrated into the circulation, in addition to a miniaturised pump and transport channels.